The field of rheumatology and therapy of autoimmune disease is facing perhaps the biggest revolution since it was established in the mid-nineteenth century. The teachings of the last 35 years are seriously challenged. The treatment pyramid for rheumatoid arthritis (RA) which seemed as stable as pyramids in the Egyptian desert is falling apart. It has been realized that RA is a more serious disease than it was thought of before. It results in higher mortality and disability as our population ages. The potentially serious changes in the joints frequently occur at the beginning of the disease. A number of rheumatologists are now suggesting more aggressive treatments, such as the use of disease-modifying antirheumatic drugs (DMARD). Effective DMARD's are yet to be found. Current disease-modifying treatments usually are less effective within two years.
Regulation of autoimmune response offers an attractive background for the introduction of new DMARD's. Every human organism has the ability to make antibodies against it own cells. Production of antibodies may play an important part in the removal of cellular remnants. Out of approximately 100,000 macromolecules present in the cell, less than 500 are autoantigens. A number of these antigens play a vital part in the synthesis of proteins. It is very important to notice that amino acyl-tRNA synthetases are among these antigens (Mathews, M. B. et al., Nature 304:177 (1983), Mathews, M. B. et al., J. Exp. Med. 160:420 (1984) and Bunn, C. C. et al., J. Exp. Med. 163:1281 (1986)). The autoimmune reaction is sustained because of persistent activation of T-lymphocytes (Sinha, A. A. et al., Science 248:1380 (1990)). The differentiation of T-cells plays a crucial part in cell tolerance. The errors in differentiation of T-cells may result in autoimmune reaction. The present inventor postulates that autoimmune disorders are the diseases of cellular differentiation. The use of differentiation inducers should lead to the remission of the disease, if such hypothesis is true.
Without wishing to bound to any proposed theory, the present inventor postulates that the human body possesses a Biochemical Defense System (BDS) (Burzynski, S. R., Internal. J. Exp. Clin. Chemother. 2:63 (1989)and Burzynski, S. R., 17th Internat. Cong. Chemother., Berlin (1991)). This system parallels the immune defense, but protects the organism against the enemy within the body. The main purpose is no longer the defense against the micro-organism, but defense against defective cells. Chemical components of this biochemical defense system are peptides, amino acid derivatives and organic acids defined as antineoplastons (Burzynski, S. R., Physiol. Chem. Phys. 8:275 (1976) and Burzynski, S. R., U.S. Pat. No. 4,470,970). The mechanism of defense is based not on destruction, but on the reprogramming of defective cells through induction of differentiation.
The research on antineoplastons began in Poland in 1967 (Burzynski, S. R., Experientia 25:490 (1969) and Burzynski, S. R., Drugs Exptl. Clin. Res. Suppl. 1 12:1 (1986)). Initially, the work concentrated on the isolation of peptides which exist in the blood of healthy people and are deficient in cancer patients. Due to the small amount of raw material available for the study, in the following years, antineoplastons were isolated from urine instead of blood. In 1980 the structure of the first antineoplaston was identified and reproduced synthetically (Burzynski, S. R. et al., Proc. 13th Internat. Cong. Chemother., Vienna, Austria 17, P.S. 12. 4. 11-4).
Antineoplastons are divided into two groups. One group contains compounds which have a wide spectrum of activity and includes Antineoplaston A1, A2, A3, A4, A5, A10, AS2-1, AS2-5. Antineoplastons A1, A2, A3, A4 and A5 contain peptides isolated from urine and Antineoplaston A10, AS2-1 and AS2-5 are the synthetic products. See e.g. U.S. Pat. Nos. 4,470,970, 4,558,057 and 4,559,325. In addition to the first group, there are antineoplastons that are active against a single specific type of neoplasm, such as Antineoplaston H, L and O. Antineoplaston A10 is the first active ingredient isolated and reproduced by synthesis. Acid hydrolysis of Antineoplaston A10 initially produces phenylacetylglutamine and phenylacetylisoglutamine. When hydrolysis is carried further, the products of reaction include phenylacetic acid, glutamic acid and ammonia. The sodium salt of phenylacetylglutamine was named Antineoplaston AS2-5 and the mixture of the sodium salts of phenylacetylglutamine and phenylacetic acid in the ratio of 1:4 was named Antineoplaston AS2-1 (Burzynski, S. R. et al., Drugs Exptl. Clin. Res. Suppl. 1 12:11 (1986)).
According to the present inventor, AS2-1 seems to induce differentiation by reducing the level of glutamine in cells and substituting glutamine with phenylacetylglutamine. Relative excess of glutamine is essential for entering S-phase of cell cycle (Zetterberg, A. et al., Cell Physiol. Chem. 108:365 (1981)). In Swiss 3T3 cells cultured and starved to quiescence, a relative excess of glutamine is necessary for approximately seven hours from the end of G.sub.o to the beginning of S phase (Zetterberg, A. et al., Cell Physiol. Chem. 108:365 (1981)).
The availability of glutamine for cells in the human organism is regulated through the well-known reaction of the conjugation of glutamine with phenylacetic acid to phenylacetylglutamine (Thierfelder, H. et al., Z. Physiol. Chem. 94:1 (1915)). Phenylacetic acid is produced in substantial amounts in the human body and over 90% is bound with glutamine to form phenylacetylglutamine (Seakins, J. W. T., Clin. Chem. Acta. 35:121 (1971)). The type of amino acid conjugated with phenylacetic acid is different for different animals and is correlated with their evolutionary status. Conjugation of glutamine seems to be specific for humans and old world monkeys (James, M. O. et al., Proc. R. Soc. Lond. B. 182:25 (1972)). Systemic administration of AS2-1 to a patient produces a relative deficiency of glutamine and introduces phenylacetyglutamine which competes with glutamine.
According to the present inventor, there are three possible mechanisms of induction of terminal differentiation by AS2-1: deviation from the genetic code, modification of DNA bases and RNA editing (Burzynski, S. R., Drugs Under Exptl. Clin. Res. 16:361 (1990)).
According to the present inventor's hypothesis, TAG codon may not represent a termination codon in certain abnormal cells. According to data published by others, certain protozoa use stop codon TAG for incorporation of glutamine into polypeptide chain (Preer, J. R. et al., Nature 314:188 (1985) and Caron, F. et al., Nature 314:185 (1988)). This allows the protein synthesis to continue through incorporation of glutamine instead of stopping at stop codon. The existence of such change in the code may explain why abnormal cells are more sensitive to relative deficiency of glutamine than normal cells. Expression of such deviation in human cells may be affected by latent infection. It is important to notice that glutaminyl-tRNA synthetase will play a vital part in this mechanism (Rould, M. A. et al., Science 246:1135 (1989)).
Another possible mechanism of action of AS2-1 is based on modification of DNA bases in abnormal cells. During the course of cellular differentiation, the tendency could exist to eliminate methylated cytosine residues, similar to the process of elimination of 5-methylcytosine in the course of evolution (Bird, A. P., Trends Genet. 3:342 (1987)). The final result is that cytosine is converted into thymine. The disturbance of the differentiation process will create a situation where such exchange will not take place. CAG and CAA codons which are instrumental in incorporation of glutamine into the protein chain will remain, instead of being changed to TAG and TAA stop codons in differentiated cells. Such situation will make abnormal cells vulnerable to relative deficiency of glutamine.
A third possibility of selective inhibition of protein synthesis by AS2-1 in abnormal cells is through RNA editing. Such process has been described in protozoa and plants, as well as in mammals (Borst, P., Annu. Rev. Biochem. 55:701 (1986), Powell, L. M. et al., Cell 50:831 (1987) and Hiesel, R. et al., Science 246:1632 (1989) and allows a change from cytosine to uracil. This will result in stop codons UAG and UAA, instead of CAG and CAA, which were responsible for incorporation of glutamine. A disturbance of such process in abnormal cells will result in persistence of CAG and CAA codons and increased incorporation of glutamine into proteins.